Serum cholinesterase test



United States Patent 3,438,866 SERUM CHOLINESTERASE TEST Adrian J.Penicnak, Corona, N.Y., assignor to Chas. Pfizer & Co., Inc., New York,N.Y., a corporation of Delaware No Drawing. Filed Mar. 21, 1967, Ser.No. 624,695 Int. Cl. C12k 1/04 US. Cl. 195-1035 5 Claims ABSTRACT OF THEDISCLOSURE Colorimetric procedure for the determination of serumcholinesterase activity in human blood utilizing 5,5-dithiobis-(Z-nitrobenzoic acid) as a color developer reagent, quinidinesulfate as an inhibitor reagent and a halide salt of ropionylthiocholineas a substrate.

Background of the invention This invention relates to a novel diagnostictest. More particularly, it relates to an improved colorimetric methodfor determining serum or plasma cholinesterase activity in human blood.The cholinesterase present in the serum sample acts onpropionylthiocholine releasing thiocholine which in turn reacts with5,S-dithiobis-(Z-nitrobenzoic acid) producing a yellow color. Thereaction is stopped by the addition of excess quinidine sulfate followedby the subsequent measurement of the optical density.

The determination of serum cholinesterase is of particular importancewhen screening individuals who may have suffered toxic effects fromorganic phosphate insecticides and similar chemicals which can result indepressed levels of enzyme activity. Serum cholinesterase determinationsare also of importance clincally as an indicator of hepatic disease orcongenital deficiency of serum cholinesterase resulting in profoundsensitivity to succinylcholine.

Summary of the invention This invention broadly comprises the steps of:

(a) Commingling a buffered aqueous solution containing about 0.01% w./v.of 5,5-dithiobis-(2-nitrobenzoic acid) having a pH of about 7.4 at 37 C.and a sample of suspect serum, the volume ratio of said solution to saidserum being approximately 200 to 1;

(b) Adding to the resulting mixture on aqueous solution containing about0.15% w./v. of a halide salt of ropionylthiocholine in an amountsuflicient to provide a volume ratio of said aqueous solution to saidserum of Ml. Serum sample 0.02 5,5'-dithiobis-(Z-nitrobenzoic acid)reagent 4.0 Propionylthiocholine reagent 0.5 Qninidine sulfate reagent1.0

Of course, it is to be understood that any equivalent volume proportionsmay be used in lieu of those above, however, the above amounts arepreferred since the total resulting volume is appropriate for thesubsequent optical density measurements.

3,438,866 Patented Apr. 15, 1969 'ice Detailed description of theinvention The herein disclosed diagnostic method determines the amountof cholinesterase activity in human serum wherein said activity ismeasured spectrophotometrically using a spectrophotometer set at awavelength of from 400 to 420 mg. The resulting optical density ismultiplied by 100 to express chloinesterase activity in units. Thenormal range for cholinesterase activity predetermined experimentally bythe subject method is from about 45 to about units. An abnormal or lowreading, that is less than 45, indicates one of the malfunctionsdescribed earlier.

Experimentally, the diagnostic procedure is carried out in the followingmanner: An 0.02 ml. sample of suspect serum is mixed with 4 ml. of an0.01% w./v. aqueous solution of 5,5-dithiobis-(Z-nitrobenzoic acid)[DTNB] having a pH of about 7.4 at 37 C. Said serum sample mustnecessarily be nonhemolyzed since hemolyzed serum may lead to falselyelevated cholinesterase activity due to the release ofacetylcholinesterase from the erythrocytes. The aforesaid DTNB isbuffered with tris(hydroymethyl) aminomethane. To this mixture is addeda slight excess.

of an aqueous solution containing about 0.15% w./v. ofropionylthiocholine iodide. Experimentally, 0.5 ml. is added.Mechanistically, the serum cholinesterase reacts with thepropionylthiochloine iodide to form thiocholine which then reacts withDTNB to form the yellow anion of S-thio-Z-nitrobenzoic acid. The serumcholinesterase activity is measured preferably after allowing theaforesaid mixture to incubate for three minutes at 37 C. The reaction isthen terminated by the addition of 1 ml. of an aqueous solutioncontaining about 0.5 w./v. of quinidine sulfate (inhibitor). Theresulting mixture is agitated thoroughly. If cuvettes are not used ascontainers for the test procedure, the solution is transferred to atypical spectrophotometer cuvette and the optical density determinedwithin 30 minutes. Experimentally, if cuvettes other than 12. ml. areused, the optical density is converted to a 12 ml. cuvette light path bymultiplying by the appropriate factor. The aforedescribed method is wellsuited to any laboratory adapted for routine determinations.

The following examples are given to more fully ill-ustrate the presentinvention. It is to be understood that these examples are forillustrative purposes only and that the invention is not meant to belimited to the specific details of the example.

Preparation of reagents (A) Buffered aqueous solution containing 0.1%w./v. 5,5'-dithiobis-(Z-nitrobenzoic acid) [DTNB].To a one liter flaskis added mg. of DTNB and 6.64 g. of NaCl. To this solution is added 650ml. of an HCl-tris mixture (prepared by combining 400 ml. of 0.1 N HCland 250 ml. of 0.2 M tris(hydroxymethyl) amino-methane (tris) solution.Without stirring, suificient distilled water is then added to give a 1liter solution. The mixture is then stirred to permit dissolution of allreagents, and adjusted to a final pH of 7.4. If below 7.4, 1 N NaOH isused whereas if the mixture is above 7.4, 1 N HCl is added.

(B) Propionylthiocholine iodide.-To approximately 600 ml. of distilledwater in 1 liter volumetric flask is added 7.5 g. ofpropionylthiocholine iodide with stirring to allow dissolution, followedby the addition of sufiicient water to give a 1 liter solution. Thissolution is dispersed into vials, each vial receiving 1 ml. of solution.Lyophilization of these vials are out at a temperature of 7080 F. Whenready to use, the vial is reconstituted with 5.0

3 m1. of distilled water resulting in a 0.15% W./v. solution (1.5mg./ml.).

(C) Quinidine sulfate.-To approximately 600 ml. of distilled Water in a1 liter volumetric flask is added 5.0 g. of quinidine sulfate withstirring to allow dissolution, followed by the addition of sufficientwater to provide a 1 liter solution.

Example I A sample of non-hemolyzed suspect serum (0.02 ml.) is combinedwith 4.0 ml. of reagent A as prepared by the procedure outlined above.If foaming occurs, the foam is allowed to dissipate before proceedingfurther. Reagent B (0.5 m1.) is then added and the reaction mixtureagitated and allowed to incubate for 3 minutes at 37 C. Reagent C (1.0ml.) is then added, the resulting mixture mixed well and the solutiontransferred to a 12 mm. spectrophotometer cuvette. The Wavelength of thespectrophotometer is set at 410 m adjusted to zero optical density witha blank, and the optical density of the sample determined.

The optical density experimentally is 0.502 which is multiplied by 100to express cholinesterase activity in units. Hence, the sample contains50 cholinesterase units.

Example II The procedure of Example I is repeated whereinpropionylthiocholine bromide is used in preparing Reagent B instead ofpropionylthiocholine iodide. Equivalent results are obtained.

Example III The procedure of Example II is repeated wherein thefollowing amounts of sample and reagents are used:

Reagent C 4 Equivalent results are obtained.

What is claimed is:

1. An improved method for determining serum cholinesterase activity inhuman blood which comprises the steps of:

(a) comminglin-g a buffered aqueous solution containing about 0.01%W./v. of 5,5-dithiobis-(2-nitrobenzoic acid) having a pH of about 7.4 at37 C. and a sample of suspect serum, the volume ratio of said solutionto said serum being approximately 200 to 1;

(b) adding to the resulting mixture an aqueous solution containing about0.15% w./v. of a halide salt of propionylthiocholine in an amountsuflicient to provide a volume ratio of said aqueous solution to saidserum of about 25 to 1; incubating and subsequently adding (c) an amountof an aqueous solution containing about 0.5% w./v. of quinidine sulfatesufficient to provide a volume ratio of said quinidine sulfate to saidserum of about to 1;

and determining cholinesterase activity spectrophotometrically bymeasuring the optical density at a wavelength of 400 to 420 III/.0.

2. The method of claim 1 wherein said buffering agent istris(hydroxymethyl) aminomethane.

3. The method of claim 1 wherein said halide salt ofpropionylthiocholine is propionylthiocholine iodide.

4. The method of claim 1 wherein said incubating period is 3 minutes at37 C.

5. The method of claim 1 wherein the amounts of said serum sample, said5,5'-dithiobis-(2-nitrobenzoic acid) solution and said quinidine sulfatesolution are 0.02 ml., 4 ml. and 1 ml., respectively.

References Cited UNITED STATES PATENTS 3,378,463 4/1968 Guilbault et a1-1035 OTHER REFERENCES Wilkinson, An Introduction to DiagnosticEnzymology, Edward Arnold Publishers, London, pp. 197-199 1962).

ALVIN E. TANENHOLTZ, Primary Examiner.

